| Osteogenic growth peptide (OGP) is highly effect to promote bone and blood-stimulating factor. It could promote bone formation, cancellous bone mineral density and fracture healing. Some studies have been found that OGP, through autocrine and paracrine way, could stimulat human and rodents osteoblasts and bone marrow stromal stem cell differentiation, alkaline phosphatase activity and matrix mineralization. In this study, the following five aspects of OGP on the cow bone marrow stromal cells and osteoblastic cells and its mechanism and characteristics of microcapsulated OGP genetically modified cells were studied.1. Preparation of dairy osteocalcin antibodyBovine osteocalcin cDNA gene was amplified from bone of bovine by RT-PCR. PCR product was cloned into PMD-18T plasmid. Sequencing analysis showed that bovine osteocalcin cDNA gene was 303 bp and encoded 100 amino acids, which completely matched with X53699 in GenBank. Through connecting three artificial single strands DNA which contain fixed-point mutated sites by Gene splicing by overlap extension PCR, we changed some rare codons to that Escherichia coli frequently used in mature bovine osteocalcin gene, then recombined the connected production into PET-32a plasmid and transformed into the competent expressive cells of E.coli BL21(DE3). Recombinant E.coli BL21 was induced by IPTG and osteocalcin fusion protein was successfully expressed, and successfully to expressed bovine BGP anti-body.2. Osteogenic actions of the osteogenic growth peptide on bovine marrow mesenchymal stromal cells in cultureThe OGP regulated marrow mesenchymal stem cells which derived from human and rodent proliferation and differentiation into osteoblasts. Whether OGP directly regulates the bovine marrow mesenchymal stem cells differentiating into osteoblasts remains unknown. In this study, we evaluated the effect of OGP on the growth and differentiation of bovine marrow mesenchymal stem cells in culture. Our results showed that OGP promoted differentiation of the bovine stem cells. OGP increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of Osteocalcin (BGP). On the other hand, OGP dose-dependently stimulated the expression of endothelial nitric oxide synthases. These results show for the first time a direct osteogenic effect of OGP on bovine marrow stromal cells in culture, which could be mediated by the induction of endothelial nitric oxide synthases.3. The stimulating effect of OGP on the proliferation, differentiation, and mineralization of osteoblastic cells from Holstein cattleThis study investigated the effect of exogenous osteogenic growth peptide on proliferation, differentiation and mineralization of osteoblastic cells from Chinese Holstein cattle. The osteoblastic cells were isolated and cultured, then identified through morphological observation and alkaline phosphatase staining methods. The effect of the different concentrations of osteogenic growth peptide on cell growth was assessed by MTT assay. Cytoplasmic alkaline phosphatase activity and osteocalcin levels were measured by colorimetric assay and radioimmunoassay, respectively.calcium nodules were observed using alizarin red S stain. The results showed that osteogenic growth peptide can significantly promote osteoblasts proliferation, and also stimulate the alkaline phosphatase activity and osteocalcin secretion.the max effect of concentration is 10-9M. when osterix expression was inhibited, the promotion of osteogenic growth peptide to alkaline phosphatase activity and osteocalcin secretion was inhibited accordingly. This revealed that osteogenic growth peptide can promote dairy osteoblast proliferation and differentiation and is mediated by up-regulating osterix.4. Responses of free radicals to subcutaneous implantation of alginate—chitosan-alginate (ACA) microcapsules in miceOur objective was to characterize the kinetics of free radicals when responding to encapsulated cell implantations in vivo. Cell viability and serum free radical, malondialdehyde and superoxide dismutase levels were evaluated at 1, 4 and 7 days after either saline injection or the implantation of empty microcapsules or encapsulated cells. Hydrogen peroxide and malondialdehyde levels showed an initial rise in the recipients of the empty microcapsules, before decreasing to the basal level. However, in rats receiving the encapsulated cells, the levels were higher at the end of study. Nitric oxide and superoxide dismutase increased after the implantation of microcapsules with or without the BHK-21 cells, but did not alter in response to the saline injection. The viability of encapsulated cells was high in vivo and some microcapsules had broken by day 7 post-implantation. These results suggest that nitric oxide played a role in the specific response to microcapsules. Free radicals increased rapidly immediately following microcapsule transplantation, but they caused only slight cellular damage before the microencapsulated cells were exposed.5. Release of OGP from microencapsulated engineered cells and proliferation characteristics in vivo and in vitroFor the study of engineered cells microcapsules in vivo and in vitro characteristics of the proliferation and secretion. Secretory of OGP and engineered cells proliferation in microcapsule cultured in vitro and in vivo was tested. Serum antioxidant enzyme activities, lipid peroxidation and nitric oxide levels in rabbit with glucocorticoid-induced osteoporosis were also assessed. The results demonstrated that OGP engineered cells microcapsules have good secretion and cell proliferation. But when transplanted into Osteoporosis model, a large number of cells in microcapsule dead. Serum free radicals in Osteoporosis model were significantly higher, but antioxidant activity decreased. The injection after anti-oxidants can significantly reduce the serum concentration of free radicals. Thus that, OGP microencapsulated genetically modified cells have the potential for the treatment of transplantation, in vivo model of osteoporosis disease transplant failure, mainly due to osteoporosis in vivo model of high levels of oxygen free radicals, leading to Microcystis necrosis or apoptosis within the cell.
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